Amino acid derivative and bromoacetyl modified peptides for the preparation of synthetic peptide polymers, conjugated peptides, and cyclic peptides

ABSTRACT

A new amino acid derivative, N.sup.α -tert-butoxycarbonyl-N e  -(N-bromoacetyl-β-alanyl)-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates and polymers. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers. Such peptide derivatives are useful in preparing potential peptide immunogens, vaccines and therapeutics, and for substances such as peptides linked to polymers, plastics, enamels and ceramics.

This application is a divisional of copending application Ser. No.07/715,650, filed On Jun. 14, 1991, now U.S. Pat. No. 5,286,846, theentire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel amino acid a derivative, N.sup.α-tert-butoxycarbonyl-N.sub.ε -(N-bromoacetyl-β-alanyl)-L-lysine (BBAL),for use in solid-phase or solution-phase peptide synthesis forintroducing a side-chain bromoacetyl group at any desired position in apeptide sequence. The bromoacetyl group is subsequently used for thepreparation of cyclic peptides, peptide conjugates and peptide polymers.Such peptide derivatives are useful in preparing potential peptideimmunogens, vaccines and therapeutics, and for substances such aspeptides linked to polymers, plastics, enamels and ceramics.

BACKGROUND OF THE INVENTION

Conjugates of synthetic peptides with proteins, other peptides, polymers(soluble/insoluble; natural/synthetic), and surfaces of specialmaterials are being employed increasingly in biomedical research andbiotechnology. Applications of peptide conjugates include thepreparation of immunogens (including synthetic vaccines) for raisingantibodies to selected portions of protein antigens (Lerner, R. A.(1982) Tapping the immunological repertoire to produce antibodies ofpredetermined specificity. Nature 299, 592-596; Zavala, F. et al, (1985)Rationale for development of a synthetic vaccine against Plasmodiumfalciparum malaria. Science 228,1436-1440; Tam, J. P. (1988) Syntheticpeptide vaccine design: Synthesis and properties of a high-densitymultiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 85,5409-5413; and Milich, D. R. (1989) Synthetic T and B cell recognitionsites. Adv. Immunol 45, 195-282), affinity adsorbents, immunoassaycomponents, and cell adhesion surfaces (Massia, S. P. et al, Covalentsurface immobilization of Arg-Gly-Asp- andTyr-Ile-Gly-Ser-Arg-containing peptides (Ser. I.D. No.: 3) to obtainwell-defined cell-adhesive substrates. Anal. Biochem. 187, 292-301; andBrandley, B. K. et al, (1988) Covalent attachment of an Arg-Gly-Aspsequence to derivatizable polyacrylamide surfaces: Support of fibroblastadhesion and long-term growth. Anal. Biochem. 172, 270-278).

The locus of attachment of a peptide to its conjugate partner may have amajor influence on the desired biological activity or performance of theconjugate (Dyrberg, T. et al, (1986) Peptides as antigens. Importance ofOrientation. J. Exp. Med. 164,1344-1349; Schaaper, W. M. M. et al,(1989) Manipulation of antipeptide immune response by varying thecoupling of the peptide with the carrier protein. Mol. Immunol. 26,81-85; and Golvano, J. et al, (1990) Polarity of immunogens:Implications for vaccine design. Eur. J. Immunol. 20, 2363-2366).Strategies for cross-linking a peptide through a single, selected locusare often complicated by the presence of more than one amino or carboxylgroup, and the need to protect (then deprotect) amino groups if acarboxyl function is to be activated. Peptides can be more or lessselectively derivatized through their N-terminal amino groups by meansof acylation reactions in slightly acidic media and/or with use ofcertain types of reagents, such as, symmetrical anhydrides (10).However, if one wishes to synthesize a peptide in order to prepare aconjugate, planning the synthesis for this purpose can prove veryadvantageous. For example, some workers have introduced a reactivecysteine residue at the desired position for heteroligation (Green, N.et al, (1982) Immunogenic structure of the influenza virushemagglutinin. Cell 28, 477-487 and Bernatowicz, M. S. et al, (1986)Preparation of peptide-protein immunogens using N-succinimidylbromoacetate as a heterobifunctional crosslinking reagent. Anal.Biochem. 155, 95-102); Drijfhout et al completed a sequence on a solidsupport with an N-terminal S-acetylmercaptoacetyl group (Drijfhout, J.W., Bloemhoff et al, (1990) Solid-phase synthesis and applications ofN-(S-acetylmercaptoacetyl) peptides. Anal. Biochem. 187, 349-354). Thedeprotected peptide was treated with hydroxylamine to remove theS-acetyl group and then joined through its N-terminal sulfhydryl groupto a conjugate partner bearing an SH-selective electrophilic function(e.g., an N-substituted maleimide or an α-haloacetyl moiety) by means ofvery stable thioether cross-links (Bernatowicz, M. S. et al (1986),Anal. Biochem. 155, 95-102, supra and Drijfhout, J. W. et al (1990),supra).

Strategy considerations may give preference to placing sulfhydryl groupson the conjugate partner (or using those already present) and couplingit to Cys peptides by means of less stable disulfide bonds. The couplingcan be effected through an activating group, such asS-(3-nitro-2-pyridinesulfenyl)(Npys), previously placed on a cysteineside chain (Bernatowicz, M. S. et al, (1986) Preparation ofBoc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine and its use forunsymmetrical disulfide bond formation. Int. J. Peptide Protein Res. 28,107-112; Drijfhout, J. W. et al, (1988) Controlled peptide-proteinconjugation by means of 3-nitro-2-pyridinesulfenylprotection-activation. Int. J. Peptide Protein Res. 32,161-166; andPonsati, B. et al, (1989) A synthetic strategy for simultaneouspurification-conjugation of antigenic peptides. Anal. Biochem. 181,389-395). A Cys(Npys) residue, introduced in solid-phase peptidesynthesis (SPPS), will remain intact during trifluoroacetic acid (TFA)or HF cleavage steps.

For an alternative approach, the peptide synthesis program could bemodified in order to introduce an SH-selective electrophile somewhere inthe sequence, again allowing use of stable thioether cross-linkages.Lindner and Robey, F. A., (1987) Automated synthesis and use ofN-chloroacetyl-modified peptides for the preparation of syntheticpeptide polymers and peptide-protein immunogens. Int. J. Peptide ProteinRes. 30, 794-800) described the incorporation of N-terminalchloroacetyl-glycylglycyl groups in the last cycle of an automated SPPS.Subsequently, Robey, F. A. and Fields, R. L. (Robey, F. A. et al, (1989)Automated synthesis of N-bromoacetyl-modified peptides for thepreparation of synthetic peptide polymers, peptide-protein conjugates,and cyclic peptides. Anal. Biochem. 177, 373-377) and Kolodny, N. andRobey, F. A. (Kolodny, N. et al, (1990) Conjugation of syntheticpeptides to proteins: Quantitation from S-carboxymethylcysteine releasedupon acid hydrolysis. Anal. Biochem. 187, 136-140) described a similarmethod for introducing the more reactive bromoacetyl group at theN-termini of peptides made by SPPS. The former method is also describedin U.S. patent application Serial No. 07/283,849 (filed Dec. 3, 1988).Using this approach, these authors have prepared many useful immunogenseither as peptide-protein conjugates or as self-polymers of peptidesthat contain both a cysteine residue and an N-terminal bromoacetylgroup.

The main limitation to the above approach is lack of flexibility inchoosing the site for an α-haloacetyl group. To our knowledge, there hasnot yet been reported a method for introducing an SH-selectivealkylating function at any desired position in a peptide that is beingsynthesized sequentially.

Accordingly, it is desired to obtain a method for introducing abromoacetyl or chloroacetyl or other haloacetyl or haloacylcross-linking function at the N- or C-terminus or at any intermediateposition of a synthetic peptide that is being prepared.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a peptidesynthesis method which may be used to overcome the above-notedlimitations.

It is a further object of the present invention to provide an amino acidderivative for introducing the bromoacetyl or other haloacetyl orhaloacyl- cross-linking function at the N- or C-terminus or at anyintermediate position of a synthetic peptide.

Another object of the present invention is to provide a method forsynthesizing the amino acid derivative N.sup.α -tert-butoxycarbonyl-N³-(N-bromoacetyl-β-alanyl)-L-lysine (BBAL).

Still, a further object of the present invention is to provide methodsfor the preparation of cyclic peptides, peptide conjugates and peptidepolymers using BBAL.

Yet a further object of the present invention is to provide methods forsynthesizing peptides that have a cross-linking handle at any selectedlocus for the purpose of preparing peptide-based components ofbiological activity modifiers, such as immunogens, immunizing epitopes,vaccines and inhibitors, as well as bioassay and affinity separationmaterials, and medical prostheses.

The foregoing objects and other are accomplished in accordance with thepresent invention, generally speaking, by providing the amino acidderivative which is a trifunctional compound having (a) haloacetyl orother haloacyl functional group; (b) a free (i.e. un-ionized) carboxylgroup, carboxylate salt, carboxylic acid active ester, acyl halide,symmetrical anhydride or mixed anhydride; and (c) a protected primary orsecondary amine, wherein the protecting group is removable, for exampleduring a cycle of stepwise synthesis in which this trifunctionalcompound is being inserted in the desired product, such as for example,N.sup.α -tert-butoxycarbonyl-N.sup.ε -(N-bromoacetyl-β-alanyl)-L-lysine(BBAL) as well as a method for synthesizing the same. The presentinvention further relates to methods for preparing various cyclicpeptides, peptide conjugates and peptide polymers using BBAL. Thepresent invention also relates to methods for synthesizing peptides thathave a cross-linking handle at any selected locus for the purpose ofpreparing peptide-based components of biological activity modifiers,bioassay and affinity separation materials, and medical prostheses.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription. All of the references cited below are incorporated hereinby reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated in the accompanying drawingswherein:

FIG. 1 is a SDS-PAGE run of a peptide polymer obtained by intermolecularcross-linking of a BBAL-containing peptide with itself through aco-existing cysteine residue;

FIG. 2 is a chromatogram of the acid hydrolyzate of the conjugate(Gly-Arg-Gly-Glu-Pro-Thr-BBAL)_(n) -BSA (SEQ I.D. No.: 2;

FIG. 3 is a chromatogram for a 1-nmol amino acid standard, spiked withβ-alanine, using the AMINO QUANT system; and

FIG. 4 is an AMINO QUANT analysis of the acid hydrolyzate from the samepeptide-BSA conjugate described with respect to FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to trifunctional compounds which include(a) a haloacetyl or other halo acyl functional group; (b) a free (i.e.un-ionized) carboxyl group, carboxylate salt, carboxylic acid activeester, acyl halide, symmetrical anhydride or mixed anhydride; and (c) aprotected primary or secondary amine, wherein the protecting group isremovable. The haloacyl functional group may be acid (acyl) chlorides,acyl bromides or acyl fluorides or possibly acyl iodides, preferablyacyl chloride and the haloacetyl group is preferably a bromoacetyl orchloroacetyl group. The trifunctional compound also contains acarboxyl-containing group (b) which may be an acyl halide, a thio ester,and acid anhydride (either a symmetric anhydride or an asymmetricanhydride), any type of ester, a free acid or carboxylate anion. Theprotected primary or secondary amine (c) is preferably protected with agroup that is nonreactive and can be removed by treatment with asubstance such as trifluoroacetic acid or a secondary amine such aspiperidine. Such protecting groups are preferably tert-butoxycarbonyl(t-BOC) which can be removed for example by treatment withtrifluoroacetic acid or 9-fluorenylmethoxy-carbonyl (Fmoc) which can beremoved by treatment with a secondary amine such as piperidine.

The present invention further relates to a new Boc-amino acidderivative, N.sup.α -tert-butoxycarbonyl-N.sup.ε-(N-bromoacetyl-β-alanyl)-L-lysine (BBAL). With these compounds, one canintroduce the bromoacetyl cross-linking function at the N- or C-terminusor at any intermediate position of a synthetic peptide that is beingprepared by solution phase methods, or by manual, semi-automated orautomated SPPS programs employing temporary N.sup.α -Boc protection andfinal HF-induced deprotection and cleavage. Coupling of these peptidesto thiol-bearing or other nucleophile-bearing carriers can be readilyaccomplished by mixing the components in a neutral or slightly alkalinebuffered medium. The ensuing peptide-carrier conjugates may bequantitatively characterized by means of the β-alanine liberated uponacid hydrolysis of a sample. If co-reactant groups are cysteinesulfhydryls, S-carboxymethylcysteine (CMC) also appears in thehydrolyzate (Kolodny, N. et al, supra). The β-alanine residue placesadditional spacing in the cross-link and appears to be a necessary partof the structure of BBAL that results in its being a stable solid thatcan be conveniently stored, weighed and dispensed for synthesizeroperations.

The trifunctional compound and BBAL in accordance with the presentinvention may be used at any cycle of a stepwise peptide synthesis inthe same manner as other N.sup.α -Boc amino acids. The trifunctionalcompound or BBAL side chain in the resulting peptide will reactselectively with sulfhydryl groups to form thioether cross-links with(a) itself, yielding cyclic peptidic molecules or linear polymers, or(b) other molecules or surfaces, forming various conjugates orbiospecifically modified surfaces. This invention provides a means forsynthesizing peptides that have a cross-linking handle at any selectedlocus for the purpose of preparing structurally peptide-based componentsof biological activity modifiers (e.g. immunogens, immunizing epitopes,vaccines, inhibitors), bioassay and affinity separation materials, andmedical prostheses.

The synthesis of a preferred example of the trifunctional compounds ofthe present invention, BBAL, is accomplished in three stages as shown inSchemes I and II. First, N-bromoacetyl-β-alanine is prepared by aprocedure similar to the ones reported by Yamada et al. (Yamada, H. etal, (1984) Nature of the binding site around and reactivity ofhistidine-15 in lysozyme. J. Biochem. 95, 503-510) and Zaitsu et al(Zaitsu, K. et al, (1987) New heterobifunctional cross-linking reagentsfor protein conjugation, N-(bromoacetamido-n-alkanoyloxy)succinimides.Chem. Pharm. Bull. 35,1991-1997) (Scheme I, first reaction). The productis adequately separated from bromoacetic acid, resulting from hydrolysisof excess bromoacetyl bromide, by a series of extractions that obviatedthe need for an adsorption chromatography step. The product is purifiedby two crystallizations from ethyl acetate-hexane, rather than fromtetrahydrofuran-isopropyl ether, and gave a melting point about 10° C.higher than that reported by Yamada et al (Yamada et al, supra). Thisacid is converted to its N-hydroxysuccinimide active ester, SBAP,(Scheme I, second reaction) by a technically facile approach whereby theproduct itself is crystallized from the reaction mixture rather than aurea by-product. Coupling is accomplished withN,N'-diisopropylcarbodiimide in 2-propanol instead of with DCC in a lesspolar solvent as is commonly done. The synthesis of BBAL is carried outconveniently and cleanly with SBAP and commercially available N.sup.ε-Boc-L-lysine (Scheme II). BBAL is a white powder which is readilystored, weighed and used with a peptide synthesizer programmed for N^(d)-Boc amino acid derivatives. ##STR1##

During the course of developing the above synthesis, alternativereagents and solvents were tried. Preliminary examination of products byNMR or MS often revealed significant exchange of Br with Cl in thebromoacetyl moiety whenever Cl was present in the system. This problemwas especially severe when bromoacetyl chloride was used, andextractions were conducted over aqueous NaCl, suggesting an ionicmechanism for the halogen exchange. All Cl-containing reagents were theneliminated from use except 1,2-dichloroethane used to crystallize BBAL,since it and dichloromethane were the only solvents that we found fromwhich the final solidification and purification of BBAL could beachieved. The Cl content of BBAL, one time crystallized fromdichloroethane, was only 0.38%, but it increased to 1.1% followingrecrystallization from the same solvent.

A simpler analogue of BBAL, N.sup.α -tert-butoxycarbonyl-N.sup.ε-bromoacetyl-L-lysine, was prepared by three different routes, eachleading to a non-crystallizable, vitreous product. The method deemedmost satisfactory for yielding a pure product was the one thatparalleled the BBAL synthesis reported above wherein SBAP wassubstituted with N-succinimidyl bromoacetate, prepared as described byBernalowicz and Matsueda (Bernatowicz, M. S. et al, (1986) Anal.Biochem. 155, 95-102, supra).

The present invention is also directed to methods for preparingsynthetic peptide analogs. For polymerizing peptides containing thetrifunctional compound BBAL, generally, a cysteine residue or other-SH-containing nucleophile should be also part of the same peptide ifcyclization is to occur at pH 6 to about 8 or 9. Above these pH's, afree amino group or other nucleophilic component could be used.Cyclization can be performed by dissolving the BBAL and -SH-containingpeptide in an aqueous buffer at pH 7-9. Typically, a buffer consistingof phosphate or bicarbonate is used. The concentration of the peptide isapproximately 1 mg/ml or less. Nonaqueous solvent such as DMF, DMSO ormethanol could be used alone or with water along with an appropriateproton scavenger such as triethylamine or diisopropylethylamine. Thecyclized peptide is purified using high performance liquidchromatography (hplc) and often times the cyclized peptide will eluteearlier than the uncyclized precursor. This is due to the diminishedavailable hydrophobic surface area in the cyclized peptide whichminimizes its interaction with the reversed phase matrix. Thecyclization is performed for anywhere from 15 min. to 24 hrs. dependingon the specific conditions used. These include temperature (typicallyroom temperature), solvent, peptide composition and solubility of thepeptide. The cyclization reaction is generally followed with hplc andwith Ellman's reagent which allows one to monitor the amount of freesulfhydryl group being consumed.

For polymerizing peptides, the same reaction conditions as stated aboveare used with one exception that the concentration of starting peptideis typically 10-50 mg/ml. Often the peptides are not soluble at thesehigh concentrations but the reaction is allowed to continue regardlessof the observed polymerization. After polymerization, the high molecularweight polymers are obtained by dialysis of the reaction mixture againstwater followed by lyophilizing the dialysate.

For conjugating trifunctional compound and BBAL peptides to anymaterial, it is necessary to react the peptide with an SH group which isa part of the material being conjugated and the solvents and pH's areessentially the same as mentioned above for cyclizing peptides. Examplesof such materials include adjuvants, sugars, peptides, lipids, proteins,functional group-bearing polymers, ceramics, glasses and silicas.Conjugating to proteins can be accomplished as detailed by Linder W. andRobey, F. A., supra and by Kolodny N. and Robey, F. A., supra or anyother way that provides a free -SH group through either cysteine orother. For example, there are several silane-derivatized materials thatcontain a free SH group on them and these can be covalently attached toglass surfaces. These allow the basic component of the glass to thenreact with a BBAL-containing peptide and the result is a thioetherlinkage between the glass surface and the peptide.

In other examples, 2-iminothiolane, succinimidyl3-(2-pyridyldithio)propionate (SPDP), and S-acetylmercaptosuccinicanhydride (SAMSA) can be used to introduce the free sulfhydroyl groupsat the position of any material where there is a free amino group. Thefree amino group reacts with these to give a material that contains afree sulfhydryl group which can then be used to react with theBBAL-containing peptides. This procedure for using 2-iminothiolane isoutlined in the above-cited reference by Linder and Robey using theN-chloroacetyl chemistries for peptides modified at the amino termini.The above are only examples and those well-versed in the art can modifythe above examples for introducing reactive SH groups into materials,organic or inorganic for use in reacting the BBAL or BBAL-containingmaterials.

The reaction of BBAL-containing peptides is strictly pH dependent; atpH's below 7 the reaction is slowed as the pH is lowered and, as such,the products can be controlled by controlling the pH.

These and other objects of the invention are accomplished by providing amethod for the preparation of peptides containing a trifunctionalcompound such as BBAL which includes the steps of coupling thetrifunctional compound or BBAL onto a resin or amino acid or amino acidderivative to form an amide linkage between the amino acid or lysinebackbone of the trifunctional compound or BBAL and the fully protectedpeptide and then deprotecting the fully protected peptide while stillpreserving the presence of the bromoacetyl or chloroacetyl group on thepeptide.

Any peptide can be derivatized using the present method. If the goal ispolymerization or cyclization of the modified peptide, a cysteine isplaced on the peptide in addition to the trifunctional compound or BBAL.

The BBAL-peptides can be prepared by a procedure which includes forminga symmetric anhydride of BBAL or an active ester of, for example,N-hydroxybenzotriazole (HOBT), reacting the anhydride or ester with anN-terminus of a peptide being synthesized at any position along the way,to form an amide linkage, and deprotecting the peptide with anhydroushydrogen fluoride or any other similar acid.

The formation of the anhydride or ester is conducted at or about 10° C.to about 30° C., preferably about 25° C.

The reaction of the BBAL-HOBT ester or BBAL symmetric anhydride isconducted at or about 0° C. to about 30° C., preferably 25° C.

The treatment with hydrogen fluoride is conducted at about -5° C/ toabout 5° C., preferably 0° C. This treatment occurs for about 10 minutesto about 3 hours, preferably about 2 hours.

BBAL and the other trifunctional compounds can be used effectively instrategies for polymerizing a great variety of synthetic peptides. Forexample, the peptides can be polymerized "tail-to-tail" if both cysteineand BBAL have been placed in the C-terminal region of the peptidemonomers, and they are subjected to conditions that favor reaction of athiol group of one molecule with a bromoacetyl moiety on another one,etc. The monomer peptides are thus joined through stable thioetherlinkages. The result of one such experiment is shown by the SDS-PAGE runpresented in FIG. 1 which is a SDS-PAGE run of a peptide polymerobtained by intermolecular cross-linking of a BBAL-containing peptidewith itself through a coexisting cysteine residue. Lane A shows themolecular weight standards; Lane B shows the synthetic peptide polymerhaving the monomer sequence,Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Val-Ile-Tyr-Cys-BBAL-NH₂(SEQ. I.D. NO.: 1). The gel was stained with Coomassie Brilliant BlueR250. Molecular weights as high as 30 kDa or more are observable in themixture of polymer sizes. Other peptides have been polymerized in likemanner to yield polymer components with molecular weights exceeding 30kDa. In general, the size distribution of the peptide polymers dependsin part on the solubility and size of the peptide monomers; the greaterthe solubility, the higher the degree of polymerization..

There appears to be a tendency for peptides to cyclize that have 4 to 6amino acid residues between the bromoacetyl-bearing and Cys residues.Therefore, it is preferred for optimal polymerization (greatest numberof repeat units) to use peptide monomers with more than 6 interstitialresidues, more preferably ones with 15 to 20 such residues.

Synthetic peptides containing BBAL, but not cysteine, are readilyconjugated with carriers bearing reactive sulfhydryl groups. Peptidecarrier conjugates are commonly used as immunogens or test antigens. Wehave employed BSA as a carrier following reduction of some of itscystine disulfide bonds with Bu₃ P in order to release multiple thiolgroups as conjugation sites (see Examples below and Kolodny and Robey,supra). Two systems of amino acid analysis were used in this study toevaluate the number of BBAL peptides conjugated to modified BSA. The"PICOTAG" (registered trademark) chromatogram of FIG. 2 clearly shows ameasurable amount of CMC from the hydrolysis ofGly-Arg-Gly-Glu-Pro-Thr-BBAL (SEQ. I.D. NO.: 2) linked to reduced BSA.FIG. 2 is a PICOTAG chromatogram of the acid hydrolyzate of theconjugate, (Gly-Arg-Gly-Glu-Pro-Thr-BBAL)_(n) -BSA (SEQ. I.D. NO.: 2).The conjugate was formed by reaction of the BBAL peptide with Bu₃P-reduced BSA as detailed under Experimental Procedures and Kolodny andRobey, supra. CMC is cleanly resolved, allowing the sensitivemeasurement of BBAL peptide covalently coupled to BSA. β-Alanine, whichalso results from hydrolysis of the conjugate, overlaps the histidinepeak and therefore cannot be assayed using the standard conditions foranalysis in the PICOTAG system. The amino acid standard chromatogramcontaining CMC in the PICOTAG system and the calculation to quantify theCMC were presented previously (Kolodny, N. and Robey. F. A., supra).

The PICOTAG amino acid analysis system could not be used to quantify theβ-alanine that is also formed from the acid hydrolysis ofBBAL-peptide-derived structures. β-Alanine elutes at the same positionas histidine (approximately 4.2-4.5 min), and thus the two componentscannot be distinguished. A value for β-alanine could be obtained byPICOTAG if the sample undergoing analysis contained no histidine.

The second amino acid analysis system that we employed was "AMINO QUANT"(registered trademark). The 1-nmol standard chromatogram, containing 1nmol of β-alanine, shown in FIG. 3 is a chromatogram for a 1-nmol aminoacid standard, spiked with β-alanine, using the AMINO QUANT system.β-Alanine is cleanly resolved from the adjacent amino acids, Thr andAla, and, thus, is used to quantitate directly the amount ofBBAL-containing peptide conjugated to a protein. The naturally occurringamino acids from the carrier protein are likely to mask all other aminoacids in the synthetic peptide. CMC, readily detected with the PICOTAGsystem, cannot be measured from a typical AMINO QUANT run because itelutes with the acidic amino acids. It is clear from examining thischromatogram that the β-alanine peak (designated Beta-ALA) is completelyresolved from the nearest amino acids (threonine and alanine). Shown inFIG. 4 is the AMINO QUANT chromatogram of the acid hydrolyzate of thesame peptide-BSA conjugate described in FIG. 2. Again, the β-alaninepeak is cleanly baseline-resolved from the other amino acids. Thus, FIG.4 demonstrates the unique versatility of conjugating peptides tocysteine residues of a protein carrier; more than one amino acid markercan be used to quantify the amount of peptide coupled to the carrierprotein.

EXAMPLES

Materials and Methods.

Reagent grade chemicals and solvents used in the synthesis were obtainedfrom Fisher Scientific (Pittsburgh, Pa.). β-Alanine andN-hydroxysuccinimide were purchased from Sigma Chemical Company (St.Louis, Mo.). The latter reagent was recrystallized once from ethylacetate. N,N'-diisopropylcarbodiimide was obtained from Aldrich ChemicalCompany (Milwaukee, Wis.); bromoacetyl bromide was ordered from Fluka(Ronkonkoma, N.Y.); N.sup.α -Boc-L-lysine was obtained from VegaBiochemicals (Tucson, Ariz.).

With the exception of BBAL, all reagents used for the automatedsynthesis of peptides were purchased from Applied Biosystems, Inc.(Foster City, Calif.). Sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE) was performed on the peptide polymers withthe gel electrophoresis system sold by Novex (Encinitas, Calif.). Thissystem provided the materials required to run gels by the method ofLaemmli (Laemmli, U. K. (1970) Cleavage of structural proteins duringthe assembly of the head of bacteriophage T4. Nature 227, 680-685). Twoamino acid analysis systems were used as described by the manufacturersfor the analyses of products that were made using BBAL: "AMINO QUANT"(registered trademark) by Hewlett Packard, Inc. (Gaithersburg, Md.), and"PICOTAG" (registered trademark) by Waters Associates (Millipore Corp.,Milford, Mass.). CMC and bovine serum albumin (BSA) were from SigmaChemical Company and tri-n-butylphosphine (Bu₃ P) was purchased fromAldrich Chemical Company.

NMR spectra for ¹ H and ¹³ C were obtained on a Varian XL200spectrometer at 200 and 50 MHz, respectively. Typically, solutions of 10mM concentration yielded spectra after the collection of 64 freeinduction decays for ¹ H (digital resolution of 0.3 Hz) and 60,000 freeinduction decays for ¹³ C (digital resolution of 1 Hz). Assignments werebased on published spectra and known substituent effects (Kalinowski, H.O. et al, (1988) Carbon-13 NMR Spectroscopy. John Wiley & Sons, NewYork).

N-Bromoacetyl-β-alanine.

A solution of β-alanine (53.5 g, 0.60 mol) in 600 mL of water was cooledto 5° C. with an ice-alcohol bath. Bromoacetyl bromide (60.0 mL as 95%pure, 0.66 mol) was added under efficient stirring at such a rate as tomaintain the temperature below 12° C. Concurrently, 5M NaOH was added ata rate needed to keep the pH near 7. These conditions were maintainedfor 45 min after completing addition of the bromoacetyl bromide. Becausethe latter reagent is a highly toxic irritant, the above operation wascarried out in a hood. The pH of the reaction mixture was then adjustedto 1.9-2.0 using 48% HBr and its volume was reduced in a rotaryevaporator to approximately 150 mL using a 60° C. bath and aspiratorvacuum. The heavy precipitate of NaBr was removed by suction filtrationand washed with approximately 15 mL of water.

The filtrate was treated with a small volume of water to dissolve newlyprecipitated NaBr and then shaken once with hexane-ethyl ether 1: 1 v+v(450 mL) , once with ethyl ether (450 mL), and 4 times with ethylacetate-ethyl ether 1:5 v+v (450 mL each time). The first upper phase(rich in bromoacetic acid) and final lower phase were discarded. Thenext 5 upper phases were pooled, filtered through Whatman #1 paper, androtary evaporated to remove solvent.

The residue was dried under vacuum and crystallized from hot ethylacetate (81 mL) by additon of hexane (about 12 mL) and cooling to 4° C.The dried product (31.4 g) was similarly recrystallized from ethylacetate (69 mL) plus hexane (15 mL) and dried under vacuum/CaCl₂. Yield,30.0 g (23.8 % of theory); mp 88.5°-91° C. [lit. 80°-81° C.]; ¹ H NMR(DMSO-d₆) δ2.43 ppm (t, 2H, α), 3.26 (q, 2H, β), 3.84 (s, 2H, α'), 8.32(br, NH), 12.20 (br, COOH); ¹³ C NMR (DMSO-d₆) δ29.31 (α'), 33.4 (α),35.2 (β), 165.91 (Ac C=O), 172.6 (Ala C=O). Anal. Calcd for C₅ H₈ BrNO₃:C, 28.59; H, 3.84; N, 6.67; Br, 38.05. Found: C, 28.67; H, 3.97; N,6.64; Br, 38.40.

Succinimidyl 3- (bromoacetamido) propionate (SBAP). To a solution ofN-bromoacetyl-β-alanine (21.00 g, 100 mol) and N-hydroxysuccinimide(13.01 g, 113 mmol) in 2-propanol (280 mL) at room temperature was added1,3-diisopropyl-carbodiimide (16.0 mL, 101 mmol). After 8-10 min, oilyprecipitation of the product began, and the walls of the container werescratched to induce crystallization. The mixture was allowed to stand 1h at room temperature and overnight at 4° C. The crystals werecollected, washed with 2-propanol (30 mL), and redissolved in 2-propanol(200 mL brought to reflux). After an overnight stand at 4° C., thecrystals were collected, washed with 2-propanol then hexane, and driedunder vacuum/CaCl₂. Yield, 22.9 g (74.6 % of theory) mp 107°-110.5° C.[lit. (23) 104°-106° C.]; ¹ H NMR (DMSO-d₆) δ2.80 ppm (s, 4H, α"), 2.82(t, 2H, α), 3.64 (q, 2H, β), 3.82 (s, 2H, α'), 7.03 (br, NH; ¹³ C NMR(CDCl₃), δ25.62 (α"), 28.67 (α'), 31.39 (α), 35.65 (β), 166.20 (Ac C=O),167.30,169.06 (C= O, s) . Anal. Cald for C₉ H₁₁ BrN₂ O₅ : C, 35.20; H,3.61; N, 9.12; Br, 26.02. Found: C., 35.74; H 3.83; N, 9.20; Br, 26.12.

N.sup.α -tert-butoxycarbonyl-N^(e) -(N-bromoacetyl-β-alanyl ) -L-lysine(BBAL). N.sup.α -Boc-L-lysine (17.73 g, 72 mmol) was ground to a finepowder and suspended in N,N-dimethylformamide (DMF) (600 mL). SBAP(18.43 g, 60.0 mmol) was added to the suspension in 5 portions at 10-minintervals. The reaction mixture was stirred for 2 h at room temperature,allowed to stand overnight at 4° C., filtered, and rotary evaporated toremove DMF (bath 30° C., pump vacuum). The residue was shaken with amixture of ethyl acetate (960 mL), 1-butanol (240 mL) and aq 0.2M KHS04(300 mL). The upper phase was shaken twice with 0.2 M KHS04 (300 and 150mL, respectively), filtered (Whatman #1 paper), and rotary evaporated toremove solvent (32° C. bath, pump vacuum). Vacuum was applied (for atleast 2 h) to purge last traces of solvent. The oily residue wasdissolved in 1,2-dichloroethane (DCE) (400 mL) by gentle warming andswirling. The solution was slowly cooled to 15°-20° C. during which timethe product initially precipitated as an oil but was induced tocrystallize by scratching. After an overnight stand (4° C.), the productwas collected, washed (3×36-mL portions of DCE), and dried invacuum/CaCl₂. Yield, 17.3 g (65.8% of theory); mp 117-122 OC. (decomp);¹ H NMR (DMSO-d₆), δ1.34 ppm (s, 9H,α"'), 1.5 (m, 6H, β, γ, δ), 2.22 (1,2H, α'), 3.02 (q, imp), 3.50 (m, ε, β', imp), 3.82 (s+m, α+α'+imp's),6.95 (d, Lys-α-NH), 7.82 (t, NH), 8.24 (t, NH); ¹³ C NMR (DMSO-d₆) δ22.96 (γ), 28.13 (CH₃), 28.60 (β), 29.40 (α"), 30.41 (α'), 34.80 (δ),35.75 (β'), 38.17 (ε), 53.36 (α), 61.32 (imp, HOCH₂ CON), 77.90[OC(CH₃)₃, 155.50 (carbamate C=O), 165.81 (BrCH₂ C=O), 169.83,174.08(C=O's). Anal. Calcd for C₁₆ H₂₈ BrN₃ O₆ : C, 43.84; H, 6.44; N, 9.59;Br, 18.23; Cl, O. Found: C, 43.79; H, 6.61; N, 9.51; Br, 18.32; C1,0.38.

Synthesis of BBAL-Containing Peptides.

The various BBAL-containing peptides were synthesized using an automatedsolid-phase peptide synthesizer (Model 430A, Applied Biosystems, Inc.,Foster City, Calif.) that is based on the original Boc/Bzl solid-phasepeptide synthesis procedures described in 1963 by R. B. Merrifield(Merrifield, R. B. (1963) Solid phase peptide synthesis. 1. Thesynthesis of a tetrapeptide. J. Amer. Chem. Soc. 85, 2149-2154). Forintroducing BBAL residues into peptides at any desired position alongthe chain, the same double coupling cycles for asparagine that arepre-programmed into the instrument were found to be most suitable whenpeptides were synthesized on a 0.5-mmol scale, the larger of the twoscales that are preprogrammed for the Model 430A. The reason for this isthat BBAL is very soluble in DMF, but spraringly soluble in CH₂ Cl₂, andthe asparagine coupling steps of the Model 430A employ1-hydroxybenzotriazole (HOBt) ester formation in DMF.

Briefly, the synthesis first involved the following: A mixture of BBALand HOBt was made by dissolving 2.0 mmol BBAL in a solution containing2.0 mmol HOBt in 4.0 mL DMF and 0.3 mL CH₂ Cl₂, The BBAL-HOBt mixturewas added to 4.0 mL of 0.5 M N,N'-dicyclohexylcarbodiimide (DCC) in CH₂Cl₂. This mixture was agitated by bubbling with N₂ over a period of atleast 30 min at 25° C. The dicyclohexylurea by-product, whichprecipitated during the formation of the BBAL-OBt ester, was filteredoff, and the ester in the filtrate was reacted with a free amine on thePAM resin to give the BBAL-PAM-coupled product. t-Boc was removed fromthe coupled BBAL in the next cycle with TFA in CH₂ Cl₂ using themanufacturers pre-programmed protocol.

Following the synthesis, the peptide was deprotected and released fromthe PAM resin using a standard. HF deprotection method as described byRobey and Fields (Robey, F. A., and Fields, R. L. (1989), supra) forpreparing bromoacetyl-containing peptides. The only scavenger used wasanisole. As mentioned previously (Robey, F. A., and Fields, R. L.(1989), supra), sulfur-containing scavengers such as thiophenol orthioanisole were avoided as a precautionary measure.

Synthesis of Peptide Polymers Using BBAL.

Peptide polymers prepared with the use of BBAL have been synthesized ascandidate immunogens. These polymerized peptides were all assembled byfollowing the same procedure detailed previously for making peptidepolymers coupled head-to-tail with cysteine at the peptide's C-terminusand bromoacetyl at the N-terminus(18). For polymers involving the use ofsynthetic peptides containing BBAL, the peptide is designed to containcysteine at any desired position and BBAL at any other position. TheBBAL, Cys-containing peptide was dissolved in 0.5M phosphate buffer, pH7.2, at a concentration of 20 mg/mL. The polymer was formed generallywithin 3 h at ambient temperatures, but we routinely allowed the mixtureto stir overnight. The mixture was then dialyzed and lyophilized asdetailed previously (Robey, F. A., and Fields, R. L. (1989), supra).

Coupling BBAL-Containing Peptides to Bovine Serum Albumin (BSA).

Synthetic peptides were coupled to BSA following the same procedureoutlined by Kolodny and Robey (Kolodny, N. et al, supra) for couplingN-terminal bromoacetyl peptides to BSA: To 30 mg BSA dissolved in 5 mLof 0.1M NaHCO₃ were added 0.2 mL of a 0.7M solution of Bu₃ P in2-propanol, and the reaction mixture was stirred for 30 min at roomtemperature. Then, 30 mg of the BBAL-containing peptide was added, andthe reaction mixture was continuously stirred for 1 h. The entire 6 mLof the conjugation reaction mixture was dialyzed for 12 h at 4° C.against a 6-L batch of 0.1M NH₄ HCO₃ and then against 3 changes of thesame solution over a 2-day period. The conjugates were then obtained inpowder form by lyophilization.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-site                                                  (B) LOCATION: 16                                                              (D) OTHER INFORMATION: /label=modifiedaa                                      /note="BBAL"                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       LysSerIleArgIleGlnArgGlyProGlyArgValIleTyrCysXaa                              15 1015                                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                                (D) OTHER INFORMATION: /label=modifiedaa                                     /note="BBAL"                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyArgGlyGluProThrXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                       (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: peptide                                                         (B) LOCATION: 1-5                                                             (D) OTHER INFORMATION: /note="cell adhesion peptide, see pg.                  2 of Specification"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TyrIleGlySerArg                                                               15                                                                        

What is claimed:
 1. A compound comprising a moiety of the formulaN.sup.α -tert-butoxycarbonyl-N.sup.α -(N-bromoacetyl-β-alanyl)-L-lysineand further comprising a peptide moiety.
 2. A trifunctional compoundcomprising:(a) a haloacetyl or other haloacyl functional group; (b) anun-ionized carboxyl group, carboxylate salt, carboxylic acid activeester, acyl halide, symmetrical anhydride or mixed anhydride; (c) aprotected primary or secondary amine, wherein the protecting group isremovable; and (d) a peptide moiety.
 3. A compound of the formulaN.sup.α -tert-butoxycarbonyl-N.sup.α -N-bromoacetyl-β-alanyl)-L-lysine,further comprising a material conjugated thereto, wherein said materialis selected from the group consisting of peptides, sugars, lipids,proteins.
 4. A trifunctional compound comprising:(a) a haloacetyl orother haloacyl functional group; (b) an un-ionized carboxyl group,carboxylate salt, carboxylic acid active ester, acyl halide, symmetricalanhydride or mixed anhydride; (c) a protected primary or secondaryamine, wherein the protecting group is removable; and further comprisinga material conjugated thereto, wherein said material is selected fromthe group consisting of peptides, sugars, lipids, and proteins.
 5. Acompound of the formula N.sup.α -tert-butoxycarbonyl-N.sup.α-(N-bromoacetyl-β-alanyl)-L-lysine, further comprising a materialconjugated thereto, wherein said material is selected from the groupconsisting of ceramics, glasses and silicas.
 6. A trifunctional compoundcomprising:(a) a haloacetyl or other haloacyl functional group; (b) anun-ionized carboxyl group, carboxylate salt, carboxylic acid activeester, acyl halide, symmetrical anhydride or mixed anhydride; (c) aprotected primary or secondary amine, wherein the protecting group isremovable; and further comprising a material conjugated thereto, whereinsaid material is selected from the group consisting of ceramics, glassesand silicas.
 7. A compound of the formula N.sup.α-tert-butoxycarbonyl-N.sup.α -(N-bromoacetyl-β-alanyl)-L-lysine, furthercomprising a functional group-bearing polymeric material conjugatedthereto.
 8. A trifunctional compound comprising:(a) a haloacetyl orother haloacyl functional group; (b) an un-ionized carboxyl group,carboxylate salt, carboxylic acid active ester, acyl halide, symmetricalanhydride or mixed anhydride; (c) a protected primary or secondaryamine, wherein the protecting group is removable; and further comprisinga functional group-bearing polymeric material conjugated thereto.